本文選題：人臍帶間充質(zhì)干細(xì)胞　切入點(diǎn)：誘導(dǎo)　出處：《河北醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：本研究在體外特定條件下，誘導(dǎo)人臍帶間充質(zhì)干細(xì)胞(humanumhilical cord mesenchymal stem cells,hUC-MSCs)向脂肪細(xì)胞定向分化，并將誘導(dǎo)后具有脂肪細(xì)胞表型的hUC-MSCs混合脂肪顆粒移植于裸鼠腹部筋膜下，研究其在脂肪移植中促進(jìn)血管再生及向脂肪細(xì)胞分化能力，為人臍帶間充質(zhì)干細(xì)胞在脂肪移植中的應(yīng)用奠定理論基礎(chǔ)。 方法：無(wú)菌條件下取得足月妊娠分娩新生兒臍帶，用剪刀剪切成約4cm的臍帶組織段，將其用無(wú)菌生理鹽水沖洗干凈以去除臍帶中殘留的血液。齒鑷祛除一條臍靜脈和兩條臍動(dòng)脈，避免血管內(nèi)皮細(xì)胞污染組織，將臍帶中華通膠組織（Wharton’s Jelly）撕裂成條索狀，將其剪成大小約1mm3的組織塊，將華通膠組織塊置于培養(yǎng)瓶中培養(yǎng)，得到hUC-MSCs，應(yīng)用流式細(xì)胞術(shù)測(cè)定其表面抗原表達(dá)情況；合用濃度為1.0×10-6mol/L地塞米松、0.5mmol/L1-甲基-3-異丁基黃嘌呤、10mg/L胰島素、0.2mmol/L吲哚美辛這四種成脂誘導(dǎo)劑定向誘導(dǎo)hUC-MSCs向脂肪細(xì)胞分化，觀察細(xì)胞分化情況并利用油紅-O免疫組化染色鑒定細(xì)胞的分化情況；取成年雄性SD大鼠腸系膜及腹股溝處脂肪,制備成體積約為0.5~1mm3的脂肪顆粒，免疫組織化學(xué)染色及掃描電鏡觀察切取的脂肪組織與剪碎離心的脂肪顆粒其細(xì)胞形態(tài)結(jié)構(gòu)、組織中血管形態(tài)及大血管含量；雄性裸鼠30只，隨機(jī)分為7天、14天、28天三組，采用同體對(duì)照的方法，左側(cè)為實(shí)驗(yàn)側(cè)，右側(cè)為對(duì)照側(cè)，將3組又分為7天實(shí)驗(yàn)組、7天對(duì)照組、14天實(shí)驗(yàn)組、14天對(duì)照組、28天實(shí)驗(yàn)組、28天對(duì)照組；用終濃度為10μmol/L的BrdU標(biāo)定的hUC-MSCs向脂肪細(xì)胞誘導(dǎo)后，將具有脂肪細(xì)胞表型的hUC-MSCs混合脂肪顆粒注射移植至裸鼠左側(cè)腹部筋膜下，相同劑量的單純脂肪顆粒注射至裸鼠右側(cè)腹部筋膜下；移植后7天、14天、28天時(shí)取出移植的脂肪組織稱重，分析同一時(shí)期實(shí)驗(yàn)組與對(duì)照組兩組之間脂肪重量差別；組織學(xué)觀察：將取出的脂肪組織進(jìn)行組織切片HE染色，對(duì)移植的脂肪組織結(jié)構(gòu)、細(xì)胞形態(tài)及微血管含量進(jìn)行觀察；免疫組化染色：利用內(nèi)皮細(xì)胞特異性標(biāo)記CD34抗體,免疫組化法顯示微血管,將不同時(shí)期脂肪移植體按照周邊區(qū)及中央?yún)^(qū)劃分,對(duì)各組微血管含量進(jìn)行定量研究。實(shí)驗(yàn)組中利用BrdU抗體，檢測(cè)經(jīng)BrdU抗原標(biāo)記的具有脂肪細(xì)胞表型的干細(xì)胞是否存在，已確定移植的干細(xì)胞是否成活，并向脂肪細(xì)胞分化。 結(jié)果： 1實(shí)驗(yàn)室前期鑒定結(jié)果證實(shí)：流式細(xì)胞技術(shù)檢測(cè)細(xì)胞陽(yáng)性表達(dá)干細(xì)胞表面標(biāo)記CD29、CD44、CD105，而造血干細(xì)胞表面標(biāo)記CD14、CD34、CD45表達(dá)呈陰性，證明培養(yǎng)獲得的細(xì)胞為純化的間充質(zhì)干細(xì)胞。 2聯(lián)合應(yīng)用地塞米松、1-甲基-3-異丁基黃嘌呤、胰島素和吲哚美辛?xí)r誘導(dǎo)hUC-MSCs生成脂肪細(xì)胞的轉(zhuǎn)化率高達(dá)90％以上，油紅-O染色陽(yáng)性,證明干細(xì)胞向脂肪細(xì)胞分化。 3hUC-MSCs對(duì)顆粒脂肪組織移植后重量變化的影響：移植400mg顆粒脂肪組織于裸鼠腹部筋膜下,移植7天、14天、28天后實(shí)驗(yàn)組移植物平均重量分別為(399.22±5.43)mg、(380.79±7.30)mg、(362.53±9.48)mg，重量維持率分別為99.8%、95.19%、90.63%。在同樣觀察時(shí)間內(nèi),對(duì)照組平均重量分別為(277.05±6.33)mg、(230.55±9.28)mg、(202.37±7.73)mg,重量維持率分別為69.29%、57.64%、50.59%。統(tǒng)計(jì)結(jié)果顯示，在相同觀察時(shí)間內(nèi),實(shí)驗(yàn)組和對(duì)照組比較差異有顯著性意義(P 0.01)。 4移植物微血管觀察：分別于各時(shí)期周邊區(qū)和中央?yún)^(qū)取4個(gè)高倍視野，計(jì)數(shù)出每個(gè)視野微血管量。染成棕色單個(gè)內(nèi)皮細(xì)胞、內(nèi)皮細(xì)胞簇均視為1個(gè)血管計(jì)數(shù)單位，計(jì)算每平方毫米面積內(nèi)微血管含量，求其均值。中央?yún)^(qū)血管密度在7天，14天，28天時(shí)，實(shí)驗(yàn)組分別為29.44±3.86個(gè)，45.74±5.07個(gè)，60.79±7.36個(gè)，對(duì)照組分別為16.83±3.13個(gè)，32.01±5.23個(gè)，44.04±6.10個(gè)。周圍區(qū)血管密度在7天，14天，28天時(shí)實(shí)驗(yàn)組分別為65.10±4.18個(gè)，89.55±6.37個(gè)，73.30±6.33個(gè)，對(duì)照組分別為47.32±5.63個(gè)，71.47±5.51個(gè)，53.42±5.96個(gè)。利用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。同一時(shí)期實(shí)驗(yàn)組與對(duì)照組中央?yún)^(qū)血管密度的比較，7天組（P＜0.01），14天組（P＜0.01），28天組（P＜0.01），同一時(shí)期實(shí)驗(yàn)組與對(duì)照組周圍區(qū)血管密度比較7天組（P＜0.01），14天組（P＜0.01），28天組（P＜0.01）,說(shuō)明同一時(shí)期無(wú)論中央?yún)^(qū)還是周邊區(qū)血管密度實(shí)驗(yàn)組都要大于對(duì)照組；同一時(shí)期實(shí)驗(yàn)組中央?yún)^(qū)與周圍區(qū)血管密度比較7天組（P＜0.01），14天組（P＜0.01），28天組（P＜0.01），同一時(shí)期對(duì)照組中央?yún)^(qū)與周圍區(qū)血管密度比較7天組（P＜0.01），14天組（P＜0.01），28天組（P＜0.01），說(shuō)明同一時(shí)期無(wú)論是實(shí)驗(yàn)組還是對(duì)照組周圍區(qū)血管密度要高于中央?yún)^(qū)血管密度。3個(gè)時(shí)期實(shí)驗(yàn)組中央?yún)^(qū)血管密度比較（P＜0.01），，周圍區(qū)血管密度比較（P＜0.01），對(duì)照組3個(gè)時(shí)期中央?yún)^(qū)血管密度比較（P＜0.01），周圍區(qū)血管密度比較（P＜0.01），說(shuō)明實(shí)驗(yàn)組與對(duì)照組3個(gè)時(shí)期周圍區(qū)與中央?yún)^(qū)血管數(shù)目差異都具有統(tǒng)計(jì)意義。從上述結(jié)果我們可以得出：隨著時(shí)間的增加，無(wú)論實(shí)驗(yàn)組還是對(duì)照組周邊區(qū)血管密度在14天達(dá)到最高峰，28天組要高于14天組，而中央?yún)^(qū)血管密度隨著時(shí)間逐漸增加。 5免疫組織化學(xué)染色檢測(cè)到經(jīng)BrdU標(biāo)定核被染成棕色的陽(yáng)性脂肪細(xì)胞，證明移植干細(xì)胞在體內(nèi)已成活，并已長(zhǎng)成成熟脂肪細(xì)胞。 結(jié)論： 1體外誘導(dǎo)的具有脂肪細(xì)胞表型的人臍帶間充質(zhì)干細(xì)胞混合脂肪顆粒移植后，部分干細(xì)胞轉(zhuǎn)化為成熟脂肪細(xì)胞，形成受區(qū)正常的、有生命力的脂肪組織。 2移植的人臍帶間充質(zhì)干細(xì)胞可能通過(guò)旁分泌的方式分泌促血管形成因子，加速移植脂肪的新生血管形成，使移植的脂肪組織快速成活，提高移植脂肪成活率。 3人臍帶間充質(zhì)干細(xì)胞幫助形成血管，而血管的形成又促進(jìn)移植脂肪的生長(zhǎng)。 4人臍帶間充質(zhì)干細(xì)胞能產(chǎn)生新生代脂肪細(xì)胞，現(xiàn)有的脂肪細(xì)胞凋亡后，新生的脂肪細(xì)胞會(huì)代之成為有活力、持續(xù)、源源不斷的生力軍。 本實(shí)驗(yàn)證實(shí)人臍帶間充質(zhì)干細(xì)胞作為種子細(xì)胞，在脂肪移植過(guò)程中發(fā)揮重要的作用。
[Abstract]:Objective: To study the in vitro specific conditions, induced by human umbilical cord mesenchymal stem cells (humanumhilical cord mesenchymal stem cells, hUC-MSCs) and differentiated to adipocyte cells, and induced with fat cell phenotype of hUC-MSCs mixed fat granule transplantation in nude mice abdominal fascia, the research in fat transplantation to promote neovascularization and to the differentiation of adipose cells, which lays the theoretical foundation for the application of human umbilical cord mesenchymal stem cells in fat transplantation.
Methods: under sterile conditions to obtain full-term newborn umbilical cord, umbilical cord segment with scissors cutting into 4cm, the sterile saline rinse to remove residual blood in umbilical cord. The tooth forceps removed a umbilical vein and two umbilical artery endothelial cells, to avoid pollution, will pass the rubber cord the organization (Wharton 's Jelly) tear into cords will cut its tissue block size is about 1mm3, the Huatong Rubber Organization block on the culture flask, hUC-MSCs, determination of the expression of surface antigens by flow cytometry; combined with concentration of 1 * 10-6mol/L 0.5mmol/L1- -3- dexamethasone, methyl isobutyl xanthine. 10mg/L 0.2mmol/L these four kinds of insulin, indomethacin induced adipogenic differentiation into adipocytes induced by hUC-MSCs agent oriented differentiation of cells was observed by oil red -O and immunohistochemical staining of cell differentiation ; adult male SD rat mesenteric and inguinal fat, preparation of fat particle volume is about 0.5~1mm3, cut the fat tissue and cut the fat particles in the centrifugal cell morphology observed by immunohistochemical staining and scanning electron microscope, vascular morphology and vascular tissue content in male; 30 nude mice were randomly divided into 7 days, 14 days, 28 days in three groups, with body control, on the left side as the experimental side, the right side of the control side, the 3 groups were divided into experimental group of 7 days, 7 days in the control group, experimental group of 14 days, 14 days in control group, experimental group of 28 days, 28 days in control group; with the final concentration of 10 mol/L BrdU hUC-MSCs calibration to fat cells after induction of hUC-MSCs mixed fat injection into nude mice with left abdominal fascia fat cell phenotype under the same dose of pure fat particles injected into nude mice right wall after transplantation; 7 Day, 14 days, remove the transplantation of adipose tissue weighing 28 days, the analysis of the experimental group and the control group during the same period between two groups of fat weight difference; histological observation: the removal of the adipose tissue HE staining of adipose tissue transplantation and the structure, micro morphology of blood cells were observed by tube; histochemical staining: the endothelial cell specific marker CD34 antibody, immunohistochemistry showed the microvessels, the fat graft according to different periods of peripheral zone and central division, quantitative study of the microvascular in every group. Using BrdU antibody in experimental group, detected by BrdU antigen labeled with fat cell phenotype of stem cells there have been determined whether the transplanted stem cells survived and differentiated into adipocytes.
Result錛

本文編號(hào)：1683776